Toll genes are associated with the determination of dorsoventral axis in the embryogenesis of Drosophilia (Cell 52, 269-279, 1988; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996), and with innate immunity detecting invading pathogens in adult body (Nature 406, 782, 2000; Nat. Immunol. 2, 675, 2001; Annu. Rev. Immunol. 20, 197, 2002). It has been clarified the Toll is a type I-transmembrane receptor having Leucine-rich repeat (LRR) in the extracellular domain, and that the intracytoplasmic domain is highly homologous with the intracytoplasmic domain of mammal-Interleukin-1 receptor (IL-1R) (Nature 351, 355-356, 1991; Annu. Rev. Cell Dev. Biol., 12, 393-416, 1996; J. Leukoc. Biol. 63, 650-657, 1998).
Recently, mammal homologue of Toll has been identified, that is the Toll Like Receptor (TLR) (Nature 388, 394-397, 1997; Proc. Natl. Acad. Sci. USA95, 588-593, 1998; Blood 91, 4020-4027, 1998; Gene 231, 59-65, 1999) and 10 members of human TLR family such as TLR2 and TLR4 have been reported so far. The role of TLR family is to recognize discrete pathogen-associated molecular patterns (PAMPs) as pattern recognition receptor (PRR) recognizing common bacterial structure, to trigger the activation of similar intracellular signaling pathway leading to the nuclear translocation of a transcription factor, NF-κB. The signaling pathway ultimately culminates in the production of inflammatory cytokines to evoke host defense responses and further evoke host defense responses to acquired immunity. Moreover, various TLR ligands are reported recently.
TLR2 recognizes a variety of bacterial components, such as peptidoglycan (PGN), bacterial tri-acylated lipoproteins, mycoplasmal di-acylated lipoproteins, and GPI anchor of Trypanosoma cruzi (Science 285, 732, 1999; Science 285, 736, 1999; J. Biol. Chem. 274, 33419, 1999; Immunity 11, 443, 1999; J. Immunol. 164. 554, 2000; Nature 401, 811, 1999; J. Immunol. 167, 416, 2001). TLR4 is essential for responses to LPS, a glycolipid specific to Gram-negative bacteria cell wall. TLR5 is reported to recognize flagellin, a protein component of bacterial flagella. Furthermore, nucleotides specific to pathogens and nucleotide analogues are also detected by TLRs. In other words, TLR 3, TLR 7 and TLR 9 participate in the recognizition of viral double stranded RNA, imidazoquinolines and bacterial DNA with unmethylated CpG motif, respectively (Nature 406, 782, 2000; Nat. Immunol. 2, 675, 2001; Annu. Rev. Immunol. 20, 197, 2002; Nat. Immunol. 3, 196, 2002).
As TLRs can form heterodimers, their ligand specificity can be further defined. Notably, TLR6 has a unique property to recognize a mycoplasmal lipoprotein by interacting with TLR2 (Proc. Natl. Acad. Sci. USA 97, 13766, 2000; Int. Innmunol. 13, 933, 2001). TLR6-deficient mice (TLR6−/−) do not respond to di-acylated mycoplasmal lipopeptides, termed macrophage-activating lipopeptide 2-kD (MALP-2), and do not produce inflammatory cytokines. On the other hand, TLR6-deficient mice respond normally to a tri-acylated bacterial lipopeptide. TLR2−/− macrophages do not respond to neither of these lipopeptides (Int. Immuno. 13, 933, 2001). That is, it becomes clear that TLR6 discriminates a subtle difference in the acylization of lipopeptides derived frommicrobial pathogens. Furthermore, these findings raise the possibility that TLR2 forms a heterodimer with a different TLR to recognize other PAMPs in the tri-acylated lipopeptides.
On the other hand, lipoproteins are produced by a variety of pathogens including mycobacteria, Gram-negative bacteria and mycoplasma species (Microbiol. Rev. 60, 316, 1996). The N-terminus acylated lipopeptide region is responsible for the immunostimulatory activity of bacterial and mycoplasmal lipoproteins. Bacterial and mycoplasmal lipoproteins differ in the degree of acylation of N-terminus cysteine. Lipoproteins of bacteria are tri-acylated, whereas those of mycoplasma are di-acylated (Trends Microbiol. 7, 493, 1999). Synthetic lipoprotein analogue consisting of a palmitoyled version of N-acyl-S-diacyl cysteine and S-diacyl cysteine mimic the immunostimulatory activity of bacterial and mycoplasmal lipoprotein, respectively (Immunobiology 177, 158, 1988; J. Exp. Med. 185, 1951, 1997).
TLR1 shows high similarity with TLR6 (Gene 231, 59, 1999). It was reported that overexpression of TLR1 inhibited the TLR2-mediated responses to modulin which are phenol-soluble proteins secreted from Staphylococcus epidermidis (J. Immunol. 166, 15, 2001). On the other hand, another report showed that TLR1 participates in the recognition of soluble factors from Neisseria meningitides (J. Immunol. 165, 7125, 2000). However, the ligand of TLR1 in vivo is yet to be clarified.
The response to bacterial components in vivo is estimated to vary upon the difference of the expression level of each TLR on the surface of the cells, but the involvement of each member of TLR family to the signaling by the stimulation of bacterial components in vivo is not yet clarified. Moreover, it was known that water-insoluble lipoprotein/lipopeptide existing in biomembranes and the like activates immunocytes. However, no protein specifically recognizing mycobacterial lipoproteins/lipopeptides was known. The object of the present invention is to provide non-human animal model non-responsive to mycobacterial lipoproteins/lipopeptides wherein the function of the gene encoding specifically mycobacterial lipoproteins/lipopeptides, useful to clarify the involvement of each member of TLR family to the signaling by stimulation of mycobacterial lipoproteins/lipopeptides, especially the in vivo function of TLR1, is deleted on its chromosome, especially a non-human animal wherein the function of TLR1 gene is deleted on its chromosome, and a method for screening substances promoting or suppressing response to mycobacterial lipoproteins/lipopeptides by using these.